CD40 ligand (CD40L or CD154) is widely recognized as one of the most important endogenous activators of the immune system. In particular, CD40L stimulates dendritic cells (DCs) to initiate strong CD8+ T cell responses. However, despite several attempts, no form of CD40L has been licensed for clinical use. To solve this problem, this project will advance the development of a highly active, soluble form of CD40L. This novel protein is made by fusing the body of Acrp30 (a natural serum protein) with the extracellular domain of CD40L. The final protein, Acrp30-CD40L or MegaCD40L, has been validated as a highly effective adjuvant for vaccine-induced CD8+ T cell responses. In this SBIR Phase 1 project, MegaCD40L will be advanced toward the clinic by developing research cell banks of cGMP CHO cells that produce human and murine MegaCD40L. The resulting proteins will be purified to homogeneity using methods that can be scaled up to industrial production. To prove the utility of this new adjuvant, MegaCD40L will be tested in a mouse vaccination system called cross-prime-short interval boost as developed by Dr. John Harty (University of Iowa), a Consultant to the project. In this vaccination protocol, mice are first vaccinated with PLGA microspheres coated with antigen followed 7 days later by boosting with antigen plus MegaCD40L plus poly(I:C). This is anticipated to generate a truly enormous antigen-specific CD8+ T cell response that would be important for vaccines against influenza, malaria, and infections caused by biodefense agents. At the conclusion of this project, MegaCD40L will be ready for BLA-enabling cGMP protein manufacturing and formal toxicity testing as the next steps toward a human trial with this exciting new vaccine adjuvant.